Mammalian development and embryonic stem cells Aryeh Warmflash KITP - morpho16 7/29/16 Outline A. Early Mammalian Development 1. Fertilization through blastocyst stage 2. Mechanisms of gastrulation 3. Some things we don’t know B. Stem cells 1. Types of embryonic stem cells 2. Basic properties 3. Molecular aspects 1. Signaling 2. Transcription factor networks 3. Epigenetic 4. Reprogramming 5. Uses of stem cells for studying development Early mammalian development Early cleavage stages Unique nature of mammalian cleavage -Cleavages are slow (12-24 hrs apart). Unique nature of mammalian cleavage -Zygotic- Cleavages are slow (12-24 hrs apart). genome is activated early (2-4 cell stage). -Lack of- Rota?onal cleavage. maternally generated prepattern - Asynchrony of cleavage. - Zygo?c genome is ac?vated early (8-cell stage in humans). Pregastrulation development ICM hypoblast trophoblast Arnold & Robertson Nat Rev Mol Cell Biol 2009 Summary of lineage decisions in the mammalian embryo Gastrulation D Shook Gastrulation D Shook Gastrulation - creating the three germ layers Mesoderm Epiblast/Ectoderm Endoderm adapted from AK Hadjantonakis Extraembryonic endoderm Arnold & Robertson Nat Rev Mol Cell Biol 2009 Molecular basis of gastrulation 1. Nodal induces its own inhibitors at the distal tip of the embryo in the distal visceral endoderm (DVE). Arnold & Robertson Nat Rev Mol Cell Biol 2009 2. DVE migrates to the anterior side Srinivas et al Development 2004 3. Presence of inhibitors on the anterior side causes restriction of Nodal to the posterior 4. A positive feedback loop of three signaling pathways maintains posterior/distal expression and is necessary for gastrulation Issues with this model 1. State of the data Brennan et al Nature 2001 -Most of our evidence is indirect from genetic knockouts Dunn et al Development 2004 2. Do we really understand how Nodal works A. Are expression patterns consistent with function? -How does it induce the DVE at the distal tip, if it is in a PD gradient -Nodal is expressed throughout the epiblast prior to gastrulation B. Does Nodal RNA —> Nodal protein —> Activity? We have no data C. Nodal gradient must be dynamic, how do cells interpret changing signals Mesoderm D. Human ≠ Mouse !! Epiblast/Ectoderm Endoderm Extraembryonic endoderm What can we do in cell culture Make patterns Study dynamics I Heemskerk What can we do in cell culture Make patterns Study dynamics I Heemskerk Play with Geometry S Chhabra Modulate dynamics B Sorre Play with Geometry S Chhabra Modulate dynamics B Sorre Stem cells come from early embryos Note mESCs and hESCs come from the embryos at same stage but represent different stages of development We are able to do this because of the unique features of early mammalian embryogenesis How can we tell if cells are pluripotent? How can we tell if cells are pluripotent? 1. Embryoid body formation 2. Teratoma formation 3. tetraploid complementation ICM-like stem cells PNAS 1981 Nature 1981 mouse embryonic stem cells on a feeder layer Grow in piled up balls of cells that mimic the ICM in vivo. Epiblast-like stem cells Science 1998 Human embryonic stem cell colony Stem cells lines can be derived from the mouse epiblast that resemble hESCs Properties of hESCs and mESCs Complex manipulations can force hESCs to revert to an ICM- like state Complex manipulations can force hESCs to revert to an ICM- like state Stem cells enabled a revolution in studying mammalian development Stem cells enabled a revolution in studying mammalian development Nature 1985 1989: the first transgenic mice to Mario R. Capecchi, Sir Martin J. Evans and Oliver Smithies "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells". Hierarchy of regulation DNA/Cytoskelton epigenetics If all signals are removed from stem cells, they differentiate to neurons Signaling requirements of stem cells Signaling requirements of stem cells LIF was the first cytokine identified to maintain pluripotency and allowed the growth of mESCs without CM or feeders but they still needed serum. What was the other signal? BMP can serve as the second signal The stem cell state can also be maintained by suppression of differentiation pathways Inhibition of: Oct4:GFP -FGFR -MEK1/2 -GSK3beta The stem cell state can also be maintained by suppression of differentiation pathways Inhibition of: Oct4:GFP -FGFR -MEK1/2 -GSK3beta But is GSK3beta inhibition really suppressing a differentiation signal? Effect of GSK3beta inhibition is equivalent to Wnt stimulation Caution: There can be discrepancies between cell culture and the embryo. Completely different signals maintain the pluripotent state of human embryonic stem cells Signaling in stem cells: summary Transcriptional networks in stem cells What genes maintain pluripotency in vivo and in vitro? Relationship between pluripotency and differentiation Pluripotency genes have complex and opposing relationships with differentiated fates How do cells exit the pluripotent state? How do cells exit the pluripotent state? Epigenetics in stem cells Repressive Activating Differentiated cells can be reprogrammed to the pluripotent state Differentiated cells can be reprogrammed to the pluripotent state To Gurdon and Yamanaka “For the discovery that mature cells can be reprogrammed to become pluripotent” SCNT can be done in human cells as well. Substates of the pluripotent state The cell cycle and differentiation Understanding early development with stem cells Understanding early development with stem cells Studying patterning with stem cells Spatial patterning in hESCs.